Pradimicin derivatives

ABSTRACT

The present invention concerns neutral sugar derivatives of pradimicins, their use as antifungal agents, methods for their preparation, and intermediates for their synthesis.

This application is a divisional of application Ser. No. 07/866,131,filed Apr. 8, 1992, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to semi-synthetic antifungal compounds,their therapeutic use, pharmaceutical compositions containing them, andmethods for their preparation. More particularly, these antifungalcompounds are derivatives of pradimicins.

2. Background Art

Pradimicins, also known as BU-3608 antibiotics, are a group ofantifungal antibiotics produced by Actinomadura hibisca sp. nov. Variouspradimicins that have been isolated from fermentation broths ofActinomadura hibisca or variants or mutants thereof, and theirstructures are depicted below: ##STR1##

Pradimicin

A: R¹ =CH₃ ; R² =CH₃ ; R³ =β- D-xylosyl

B: R¹ =CH₃ ; R² =CH₃ ; R³ =H

C: R¹ =CH₃ ; R² =H; R³ =β- D-xylosyl

D: R¹ =H; R² =CH₃ ; R³ =β-D-xylosyl

E: R¹ =H; R² =H; R³ =β-D-xylosyl

FA-1: R¹ =Ch₂ OH; R² =CH₃ ; R³ =β-D-xylosyl

FA-2: R¹ =CH₂ OH; R² =H; R³ =β-D-xylosyl Pradimicin A was reported asBMY-28567 in Abstract No. 984 of the 27th Interscience Conference onAntimicrobial Agents and Chemotherapy, Oct. 4-7, 1987, New York, N.Y.

Pradimicins A, B, C, and the aglycone are disclosed in European PatentApplication No. 277,621.

Pradimicins D and E, and their respective desxylosyl analogs aredisclosed in our co-pending application, U.S. Ser. No. 203,776, filedJun. 7, 1988.

Pradimicins FA-1 and FA-2, their respective desxylosyl derivatives,N-alkyl derivatives thereof, and the aglycone are disclosed in ourco-pending application, U.S. Ser. No. 269,821, filed Nov. 10, 1988.

Two compounds, known as benanomicins A and B, were reported in J.Antibiot., 1988, 41 (6):807-811, and ibid, 41 (8):1019-1028. BenanomicinB appears to be identical to pradimicin C, whereas benanomicin A has thefollowing structure II: ##STR2## Desxylosyl benanomicin B was alsodisclosed but desxylosyl benanomicin A was not.

SUMMARY OF THE INVENTION

The present invention provides compounds of formula III ##STR3## whereinR¹ is selected from the group consisting of hydrogen, methyl, andhydroxymethyl, and when R¹ is methyl or hydroxymethyl, the resultingamino acid residue has the D-configuration; and R² is selected fromhydrogen or β-D-xylosyl with the proviso that when R¹ is methyl R² ishydrogen; or a pharmaceutically acceptable salt thereof.

Another aspect of the present invention provides intermediates offormula IV ##STR4## wherein R^(a) is H, methyl, or hydroxymethyl, andwhen R^(a) is methyl or bydroxymethyl the resulting amino acid has theD-configuration; R^(c) is C₁₋₅ alkyl; and R^(d) is C₁₋₅ alkanoyl.Compounds of formula IV are useful intermediates in the preparation ofcompound of formula III as well as other pradimicin derivatives. Alsoprovided is a method for the preparation of IV which comprises reactinga pradimicin aglycone ester with an acyl halide in the presence of aphase transfer catalyst.

A further aspect of the present invention provides compounds of formulasV and VI ##STR5## wherein R^(a) is H, methyl, or hydroxymethyl, and whenR^(a) is methyl or e hydroxymethyl, the resulting amino acid has theD-configuration; R^(b) is H or β-D-xylosyl; or a salt thereof, or anester thereof. Compounds of formulas V and VI are useful intermediatesfor the preparation of compounds of formula III.

Yet a further aspect of the present invention provides a process forpreparing a compound of formula VII ##STR6## wherein R^(a) and R^(b) areas previously defined, or a pharmaceutically acceptable salt thereof,which comprises the steps of (a) reacting a pradimicin having a primaryamino group with 3,5-di-t-butyl-1,2-benzoquinone in an inert organicsolvent to provide the corresponding imine; (b) converting the imineinto the corresponding ketone in the presence of an acid catalyst; (c)reducing the ketone to the hydroxyl group; and (d) separating theisomers.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, unless indicated otherwise explicitly or by context, theterm "pharmaceutically acceptable salt" refers to salts formed withinorganic or organic bases and includes, but is not limited to, sodium,potassium, lithium, calcium, magnesium, ammonium, and trialkylammoniumsalts; "pradimicin" represents a member of the naturally occurringpradimicins, their desxylosyl derivatives, and salts thereof."Pradimicin aglycone" refers to a compound having the formula VIII##STR7## wherein R^(a) is as defined under formula IV.

The pradimicin starting materials and methods for their production aredisclosed in our co-pending applications, U.S. Ser. No. 115,273, filedNov. 2, 1987, U.S. Ser. No. 203,776, filed Jun. 7, 1988, U.S. Ser. No.221,144, filed Jul. 19, 1988, and U.S. Ser. No. 269,821, filed Nov. 10,1988. The disclosures contained in these applications are herebyincorporated by reference. The pradimicins may be used as the free base,acid or base addition salts, the internal salt, or esters of thecarboxylic group, depending on the particular reaction conditions. Basesalts may be, e.g., sodium, potassium, lithium, calcium, magnesium,ammonium, and trialkylammonium salts; acid addition salts may be, e.g.,hydrochloride, sulfate, nitrate, and the like; carboxylic acid ester maybe a lower alkyl ester, e.g. methyl, ethyl, and isopropyl or acycloalkyl ester, e.g., cyclohexyl, phenyl, or benzyl ester.

Compounds of formula III may be prepared by two general methods: (1)glycosidation of an 1-O-acylated pradimicin aglycone ester with theappropriate monosaccharide or disaccharide; or (2) conversion of thesugar amino group of a pradimicin into a keto group followed byreduction to a hydroxyl group. These two approaches are illustratedschematically and discussed in detail below. ##STR8##

In Scheme I, R¹ and R² are as previously defined under formula III.Pradimicin aglycone esters of formula IX are generally insoluble orpoorly soluble in organic solvents such as methylene chloride,chloroform, dichloroethane, and dioxane making it inconvenient asstarting material for direct glycosidation with the desired sugar. Thusone aspect of the present invention is the conversion of IX into acorresponding solvent soluble acylated derivative. The pradimicinaglycone ester IX is acylated under phase transfer conditions using asacylating agent such as an acyl halide. Suitable acyl halides are forexample acetyl chloride and propionyl chloride. The reaction isconducted in an inert organic solvent such as methylene chloride,tetrahydrofuran, ether, and dioxane and toluene. The reaction mixtureincludes a base in solid form; suitable bases include sodium hydroxide,potassium hydroxide, sodium bicarbonate, sodium carbonate and the like.The phase transfer catalyst may be for example tetrabutylammoniumhydrogen sulfate, tetrabutylammonium dihydrogen phosphate, as well asother reagents that can bring the pradimicin reactant into the samephase as the acylating reagent. The reaction may be carried out attemperatures ranging from about -50° C. to about 50° C., but preferablyit is carried out at room temperature. The reaction time may range fromseveral minutes to several hours. In a preferred embodiment theacylation is effected in an organic solvent using acetyl chloride in thepresence of tetrabutylammonium hydrogen sulfate (TBAH) and powderedsodium hydroxide; the reaction using these reagents generally takes lessthan one hour to complete at room temperature. Phase transfer catalyzedacylation using TBAH/NaOH/organic solvent is described by Illi, V.O. inTet. Lett., 1979, 2431-2432. Using the procedure provided herein above,the phenolic hydroxyl group at the 1-position is preferentially acylatedover the aliphatic hydroxyl groups and the phenolic hydroxyl groups atthe 9- and 14-positions.

The acylated pradimicin aglycone ester X is then glycosylated underKoenigs-Knorr conditions. Typically, a peracylated glycosyl halide suchas peracetylated fucosyl bromide or peracetylated3-O-(β-D-xylopyranosyl) fucosyl bromide is used, and the reaction iscarried out in an inert organic solvent such as methylene chloride,chloroform, 1,2-dichloroethane, dioxane, and the like, under anhydrousconditions and in the presence of a silver or mercuric salt such asmercuric cyanide and mercuric bromide. Anhydrous conditions may bemaintained by including in the reaction mixture a dehydrating agent suchas molecular sieves. Glycosylation is preferably performed at anelevated temperature for a period sufficient to substantially convertthe aglycone into the glycoside. The reaction between 1-O-acetylatedpradimicin A aglycone methyl ester and fucosyl bromide at about 80° C.is usually complete in two hours or less. The various ester linkages arethen hydrolyzed using conventional methods to remove the phenolic andsugar acyl groups, as well as the amino acid ester group. A suitablemethod is, e.g., base-catalyzed saponification at room temperature. Theglycosidation generally results in a mixture of regioisomers andanomers, including 5-O-α-, 5-O-β-, and 6-O-β-glycosylated products. Theindividual components may be separated using techniques well known inthe art, such as column chromatography, and may be done before or afterthe removal of the protecting groups.

It will be appreciated that 1-O-acylated pradimicin aglycone esters maybe used to prepare pradimicin compounds other than the ones illustratedin Scheme I if the appropriate sugar is used. ##STR9##

In the above Scheme, R¹ and R² are as defined previously under formulaIII; R^(2') is H, β-D-xylosyl; C₁₋₅ alkanoyl, preferably acetyl; orperacylated, preferably peracetylated β-D-xylosyl; R³ and R⁴ areindependently H or methyl; and R⁵ is H or acetyl A variety of methodshave been reported in the art for converting an amine into a carbonylcompound. For example, primary amines can be so transformed by treatmentwith a reagent, such as benzothiazole-2-carboxaldehyde or3,5-di-t-butyl-1,2-benzoquinone, to give the imine which is thenhydrolyzed to the corresponding carbonyl compound. Primary, secondary,and tertiary amines can be directly oxidized to the correspondingcarbonyl compounds with, e.g., manganese oxide or neutral permanganate.Tertiary amines may be oxidized with, e.g., m-chloroperbenzoic acid toits amine oxide which, in turn, is converted to the carbonyl compound bytreatment with, e.g., trifluoroacetic anhydride. Under certain reactionconditions, e.g. oxidizing conditions, it may be desirable to protectnon-reacting functional groups on the pradimicin starting material, suchas the alcoholic and phenolic OH groups; the protection and deprotectionof these functional groups are well within the skills of a person ofordinary skill in the art. Reduction of the carbonyl may be effectedusing a reducing agent such as sodium borohydride. The reduction is notstereospecific and results in a mixture of products where the carbonylderived hydroxyl group is in either the axial or the equatorialposition. The mixture may be separated by chromatography. In ourexperience, compounds of the present invention may be prepared via theimine generated by treatment of a pradimicin having a primary aminegroup with 3,5-di-t-butyl-1,2-benzoquinone. This procedure isillustrated in Scheme III and will be further elaborated below with theunderstanding that the preparation of compounds of the invention is notlimited to the method particularly exemplified. ##STR10## (i)3,5-di-t-butyl-1,2-benzoquinone, NEt₃ in MeOH; (ii) HCO₂ H/MeOH; (iii)NaBH₄.

In the above Scheme, R¹ and R² are as defined previously under FormulaIII. To elaborate on the above scheme, pradimicin is first reacted with3,5-di-tert-butyl-1,2-benzoquinone to convert the primary amino group onthe sugar moiety to the corresponding 2-hydroxy-3,5-di-tert-butylphenylSchiff base (XIV). The reaction is carried out in solution using areaction inert solvent, such as a lower alkanol, preferably methanol. Atertiary amine base, such as triethylamine, is preferably included inthe reaction mixture when an acid addition salt of pradimicin is used asthe starting material. The temperature of the reaction is not criticaland the reaction may be conveniently conducted at ambient temperature.In general, the reaction takes from about 20 minutes to several hours.The imine thus obtained is hydrolyzed in the presence of an acid toyield the ketone (XV). The acid is not particularly restricted and maybe an inorganic acid or an organic acid, such as formic, acetic, oxalicacid, and the like. The hydrolysis may be carried out in a loweralkanol, such as methanol, at a temperature ranging from roomtemperature to the refluxing temperature of the reaction solution. Theketone is then reduced to the alcohol by a conventional reducing agent;a suitable agent is, for example, sodium borohydride. The reductionusing sodium borohydride is preferably carried out at a reducedtemperature, for example, from about -10° C. to about 10° C. in anaqueous or alcoholic solution. The product of the reduction is a mixtureof axial and equatorial hydroxy compounds which are separable bychromatography for example on a C₁₈ column.

It will be noted that the methods described herein for synthesizing thenovel compounds of the present invention are also applicable forpreparing the known compound benanomicin A when the appropriate startingmaterials are used.

BIOLOGICAL PROPERTIES

The minimum inhibitory concentrations (MICs) of representative compoundsof the present invention against 14 fungi were determined by serial agardilution method using Sabouraud dextrose agar (pH 7.0). The inoculumsize of the test organism was adjusted to 10⁶ cells/ml, andapproximately 0.003 ml of fungal suspension was applied to the surfaceof agar plates containing the test antibiotics. After the plates hadbeen incubated for 40 hours at 28° C., the lowest concentration ofantibiotic causing virtually complete inhibition of fungal growth wasdetermined as the MIC. The results are summarized in Table I.

                  TABLE I                                                         ______________________________________                                        In Vitro Antifungal Actvity                                                   Test Organisms      Ex. 1    Ex. 2  Ex. 3                                     ______________________________________                                        Candida albicans                                                                             IAM4888  25.0     12.5 12.5                                    Candida albicans                                                                             A9540    25.0     12.5 12.5                                    Cryptococcus neoformans                                                                      D49      50.0     12.5 12.5                                    Cryptococcus neoformans                                                                      IAM4514  50.0     12.5 6.3                                     Aspergillus fumigatus                                                                        IAM2530  >50.0    25.0 25.0                                    Aspergillus fumigatus                                                                        IAM2034  >50.0    50.0 25.0                                    Fusarium moniliforme                                                                         A2284    >50.0    >50.0                                                                              >50.0                                   Trichophyton mentagrophytes                                                                  D155     >50.0    12.5 50.0                                    Trichophyton mentagrophytes                                                                  #4329    >50.0    12.5 50.0                                    Sporothrix schenckii                                                                         IFO8158  >50.0    25.0 12.5                                    Aspergillus flavus                                                                           FA21436  >50.0    >50.0                                                                              >50.0                                   Blastomyces dermatitidis                                                                     D40      >50.0    >50.0                                                                              50.0                                    Petriellidium boydii                                                                         IFO8078  ND       >50.0                                                                              >50.0                                   Mucor spinosus IFO5317  >50.0    >50.0                                                                              50.0                                    ______________________________________                                    

The in vivo activity of compound of Example 1 was tested against Candidaalbicans A9540 infection in mice. Test organisms were cultured for 18hours at 28° C. in YGP medium (yeast extract, glucose, peptone, K₂ HPO₄,MgSO₄) and then suspended in saline. Male ICR mice weighing 20 to 24 gwere infected intravenously with about 10 times the median lethal doseof the test fungus. The antibiotic at various dose levels wasadministered to groups of 5 mice each intravenously just after thefungal infection. The dose that protects 50% of the animals frominfection (PD₅₀, mg/kg) was calculated from survival rates recorded onthe 20th day after the fungal challenge. All control animals died within7 to 15 days after infection. Compound of Example 1 showed nosignificant in vivo activity at 50 mg/kg by a single intravenousinjection.

For treatment of fungal infections in animals and human beings, theantibiotics of the present invention may be given in an antifungallyeffective amount by any accepted routes of administration; theseinclude, but are not limited to, intravenous, intramuscular, oral,intranasal, and for superficial infections, topical administration.Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, or emulsions. They may also bemanufactured in the form of sterile solid compositions which can bedissolved in sterile water, physiological saline, or some other sterileinjectable medium immediately before use. Oral formulation may be in theform of tablets, gelatin capsules, powders, lozenges, syrups, and thelike. For topical administration, the compound may be incorporated intolotions, ointments, gels, creams, salves, tinctures, and the like. Unitdosage forms may be prepared using methods generally known to thoseskilled in the art of pharmaceutical formulations.

It will be appreciated that, when treating a host infected with a fungussusceptible to the antibiotics of this invention, the actual preferredroute of administration and dosage used will be at the discretion of theattending clinician skilled in the treatment of fungal infections andwill vary according to the causative organism, its sensitivity to theantibiotic, severity and site of the infection, and patientcharacteristics, such as age, body weight, rate of excretion, concurrentmedications, and general physical condition.

The following examples are illustrative without limiting the scope ofthe present invention. The structures of all the compounds prepared inthe following examples are given at the end of the Examples section.

Example 1. Preparation of 4'-deamino-4'-hydroxy pradimicin B (XIX)

(a) To a suspension of pradimicin A aglycone methyl ester (IX, R¹ =CH₃ ;154 mg, 0.27 mmol) in dry dioxane (3 ml) were sequentially addedtetrabutylammonium hydrogen sulfate (103 mg, 0.30 mmol), powdered NaOH(85 mg, 2.12 mmol), and 1M solution of acetyl chloride in dry dioxane(1.06 ml). The mixture was stirred at room temperature for 30 minutesunder argon atmosphere, and the insoluble matters were removed byfiltration and washed with dioxane. The filtrate and washings werecombined and evaporated to dryness, and the residue was chromatographedon silica gel (40 g) using chloroform/methanol=20/1 as eluant to afford1-O-acetylated pradimicin A aglycone methyl ester (XVI, 69 mg, 42%) asorange solid. MP 220° C. (dec.).

IR ν_(max) (KBr) cm⁻¹ 1749, 1612.

UV λ_(max) (CH₃ CN) nm (ε) 288 (25600), 448 (10300).

¹ H NMR (DMSO-d₆) δ 1.33 (3H, d, J=7.3 Hz, 17-CH₃), 2.02 (3H, s, OAc),2.37 (3H, s, 3-CH₃), 3.67 (3H, s, COOCH₃), 3.95 (3H, s, 11-OCH₃), 4.22and 4.29 (each 1H, m, J₅,6 =11.1 Hz, 5 and 6-H), 4.41 1H, dq, J₁₇,NH=6.9 Hz, 17-H), 6.13 and 6.30 (each 1H, brs, 5 and 6-OH), 6.93 (1H, d,J₁₀,12 =2.4 Hz, 10-H), 7.30 (1H) d, 12-H), 7.46 (1H, s, 4-H), 8.07 (1H,s, 7-H), 8.77 (1H, d, NH), 12.83 (1H, s, 9-OH) and 13.37 (1H, s, 14-OH).

(b) To a solution of 1-O-acetylated pradimicin A aglycone methyl ester(73 mg, 0.12 mmol) in absolute chloroform (4 ml) were added powderedmolecular sieves 3A (740 mg), Hg(CN)₂ (271 mg, 1.07 mmol), and HgBr₂(121 mg, 0.34 mmol). The mixture was stirred at room temperature for 2hours, and tri-O-acetyl-D-fucosyl bromide prepared fromtetra-O-acetyl-D-fucose (133 mg, 0.40 mmol) and 30% HBr-AcOH (1.3 ml)according to the reported procedure by M. Takai, et al., J. Med. Chem23, 549 (1980)! was added. The mixture was heated at 80° C. for 1.5hours and then filtered off and washed with chloroform. The filtrate andwashings were combined, washed with water then saturated aqueous NaC1,and dried over Na₂ SO₄. The solvent was evaporated, and the residualsyrup was chromatographed on silica gel (20 g) using toluene/ethylacetate=1/1, and chloroform/methanol=20/1, successively, as eluants toafford the glycosidated product as a mixture of several components (43mg, Y:41%).

IR ν_(max) (KBr) cm⁻¹ 1751, 1623.

UV λ_(max) (CH₃ CN) nm (E₁ cm^(1%)) 278 (177), 494 (71).

(c) A crude sample obtained above (38 mg) was treated with 1N NaOH (1.2ml) in methanol (6 ml) at room temperature for 2 hours. The mixture wasadjusted to pH 4 with 1N HC1 and then evaporated to dryness. The residuewas chromatographed on a C₁₈ column using acetonitrile/phosphate buffer(pH 3.5)=35/65 as eluant to afford 3 fractions. Each fraction was madealkaline with 1N NaOH and then placed on a C₁₈ column, washed with H₂ O,eluted with 50% aqueous acetonitrile, and lyophilized to afford thefollowing fractions as sodium salt.

Fraction 1: 4'-Deamino-4'-hydroxy pradimicin B α-anomer (XVII, 6 mg,19%). MP>230° C.

IR ν_(max) (KBr) cm⁻¹ 1618.

UV λ_(max) (1/100N NaOH) nm (ε) 319 (7000), 499 (6800).

¹ H NMR (DMSO-d₆) δ 1.08 (3H, d, J_(5'),Me =6.4 Hz, 5'-Me), 1.33 (3H, d,J₁₇,Me =7.3 Hz, 17-Me), 2.26 (3H, s, 3-Me), ca. 3.50 (2H, m, 3',4'-H) ,ca. 3.65 (1H, m, 2'-H), 3.91 (3H, s, 11-OMe), 4.12 (1H, q, 5'-H), 4.30(1H, d, J₅,6 =9.0 Hz, 5-H), ca. 4.30 (1H, m (q after addition of D₂ O ),17-H), 4.43 (1H, dd, J₆,OH =3.9 Hz, 6-H), ca. 4.5 (1H, m, OH), ca. 4.6(2H, m, OH×2), 4.81 (1H, d, J_(1'),2' =2.6 Hz, 1'-H), 5.62 (1H, d,6-OH), 6.71 (1H, d, J₁₀,12 =2.6 Hz, 10-H), ca. 7.05 (1H, brs, 4-H), 7.12(1H, d, 12-H), and 7.63 (1H, s, 7-H).

Fraction 2: 6-P-(β-D-fucopyranosyl) pradimicin A aglycone (XVIII, 10 mg,32%). MP>230° C.

IR ν_(max) (KBr) cm⁻¹ 1617.

UV λ_(max) (1/100N NaOH) nm (ε) 316 (11200), 498 (10300).

¹ H NMR (DMSO-d₆ +D₂ O ) δ 1.14 (3H, d, J_(5'), Me =6.0Hz, 5'-Me), 1.29(3H, d, J₁₇,Me =6.8 Hz, 17-Me), 2.23 (3H, s, 3-Me), 3.43 (1H, d,J_(3'),4' =3.9 Hz, 4'-H), 3.48 (1H, dd, J_(2'),3' =9.0 Hz, 3'-H), 3.52(1H, d, J_(1'),2' =7.3 Hz, 2'-H), 3.56 (1H, q, 5'-H), 3.86 (1H, q,17-H), 3.89 (3H, s, 11-OMe), 4.38 (1H, d, J₅,6 =11.1 Hz, 6-H), 4.42 (1H,d, 5-H; simplified after addition of D₂ O), 4.58 (1H, d, 1'-H), 6.70(1H, br d, 10-H), 6.83 (1H, s, 4-H), 7.13 (1H, br d, 12-H), and 7.78(1H, s, 7-H).

Fraction 3: 4'-Deamino-4'-hydroxy pradimicin B (XIX, 2 mg, 6%). MP>230°C.

IR ν_(max) (KBr) cm⁻¹ 3396, 1620.

UV λ_(max) (1/100N-NaOH) nm (ε) 319 (10700), 498 (10400).

¹ H NMR (DMSO-d₆ +D₂ O ) δ 1.14 (3H, d, J_(5'),Me =6.4 Hz, 5'-Me), 1.31(3H, d, J₁₇,Me =6.8 Hz, 17-Me), 2.23 (3H, s, 3-Me), 3.39 (1H, dd, 3'-H),3.44 (1H, d, J_(3'),4' =3.4 Hz, 4'-H), 3.53 (1H, dd, J_(1'),2' =8.1 Hz,J_(2'),3' =9.0 Hz, 2'-H), 3.57 (1H, q, 5'-H), 3.85 (1H, q, 17-H), 3.91(3H, s, 11-OMe), 4.37 (1H, d, J₅,6 =11.1 Hz, 5-H), 4.46 (1H, d, 6-H;simplified after addition of D₂ O), 4.53 (1H, d, 1'-H), 6.66 (1H, br d,10-H), 6.99 (1H, s, 4-H), 7.15 (1H, br d, 12-H), and 7.68 (1H, s, 7-H).

Mass (HR-FAB) m/z 695.1832; Calcd. for C₃₄ H₃₃ NO₁₅ : 695.1813

Example 2. Preparation of 4'-deamino-4'-hydroxy pradimicin E (XXII)

(a) Triethylamine (0.15 ml, 1.05 mol) was added to a mixture ofpradimicin E HC1 (150 mg, 0.18 mmol), and3,5-di-tert-butyl-1,2-benzoquinone (110 mg, 0.5 mmol) in dry methanol(4.5 ml). The mixture was stirred overnight and concentrated underreduced pressure. To the residue were added ethyl acetate (5 ml) and aq.saturated NaHCO₃ (2 ml), and the mixture was stirred for 30 minutes atroom temperature to precipitate the sodium salt of4'-(3,5-di-t-butyl-2-hydroxy)phenyl imine of pradimicin E (XX, 205 mg).

IR ν_(max) (KBr) cm⁻¹ 1617, 1258, 1078.

UV λ_(max) (methanol) nm (E₁ cm^(1%)): 284 (225), 495 (91).

¹ H NMR (DMSO-d₆) δ:0.95 (3H, d, J=7 Hz, 5'-CH₃), 1.21 (9H, s t-Bu),1.24 (9H, s, t-Bu), 2.23 (3H, s, 3-CH₃), 4.81 (1H, d, J=8 Hz, 1'-H),5.15 (2H, br)*, 5.99 (1H, s)*, 6.43 (1H, d, J=2 Hz, phenyl-H), 6.51 (1H,d, J=2 Hz, phenyl-H), 6.70 (1H, br, 10-H), 6.90 (1H, s, 4-H), 7.10 (1H,br, 12-H), 7.70 (1H, s, 7-H), 15.02 (1H, s)*.

* Disappeared upon addition of D₂ O.

(b) A mixture of the product obtained in step (a) (200 mg, 0.19 mmol),formic acid (2.5 ml), and methanol (2.5 ml) was heated at 60° C. for 1.5hours. The reaction mixture was concentrated under reduced pressure, andthe residue was chromatographed on a column of C18 silica gel (20×200mm). The column was eluted with water and then with 80% acetonitrile.The acetonitrile fractions were checked with HPLC, and the desiredfractions were combined and concentrated to leave an aqueous residue,which was freeze-dried to give 4'-deamino-4'-oxo pradimicin E (XXI, 84mg, 89%) as an amorphous powder.

IR ν_(max) (KBr) cm⁻¹ 1620, 1260, 1084.

UV λ_(max) (0.01N NaOH) nm (ε): 319 (11600), 497 (10700).

¹ H NMR (DMSO-d₆) δ:3.88 (3H, s, OCH₃), 6.69 (1H, s, 10-H), 6.90 (1H, s,4-H), 7.09 (1H, s, 12-H), 7.72 (1H, s, 7-H).

(c) To a stirred mixture of the product obtained in step (b) (90 mg,0.11 mmol), 1N NaOH (0.25 ml), and water (9 ml) was added an aqueoussolution of 0.1M sodium borohydride (0.4 ml) at 5° C. The mixture wasstirred for 30 minutes at the same temperature and acidified with 1N H₂SO₄ to destroy the reagent. The mixture was adjusted to pH 8 with NAHCO₃and chromatographed on a column of C18 silica gel (40×330 mm, 5%acetonitrile), followed by preparative HPLC (System 500 (Waters), 15%acetonitrile) to give 3 fractions--a faster moving fraction containingthe equatorial isomer, a slower moving fraction containing the axialisomer, and a fraction containing a mixture of both isomers. Eachfraction was concentrated to a small volume, acidified with 1N H₂ SO₄,and subjected to a short column of C18 silica gel. The column was washedwith water and eluted with 80% acetonitrile. The eluate was concentratedto a small volume and lyophilized. The 3 fractions afforded4'-deamino-4'-hydroxy pradimicin E axial isomer (XXII, 7.5 mg, 8%), theequatorial isomer (XXIII, 4.8 mg, 5%), and a mixture thereof (8.2 mg,9%).

4'-Deamino-4'-hydroxy pradimicin E (axial isomer, XXII)

MP:>220° C. (grad. dec.)

UV λ_(max) (0.01N NaOH) nm (E₁ cm^(1%)): 236 (317), 319 (151), 496(140).

IR ν_(max) (KBr) cm⁻¹ 3288, 2921, 1728, 1628, 1607.

¹ H NMR (DMSO-d₆) δ:1.1 (3H, d, J=6.4 Hz, 5'-CH₃), 2.33 (3H, s, 3-CH₃),3.91 (2H, d, J=6.06 Hz, NH-CH₂ -), 3.95 (3H, s, 11-OCH₃), 4.40 (1H, d,J=7.3 Hz, 1"-H), 4.64 (1H, d, J=7.7 Hz, 1'-H), 6.89 (1H, s, 10-H), 7.11(1H, s, 4-H), 7.25 (1H, s, 12-H), 7.98 (1H, s, 7-H).

HPLC*: Retention time 9.8 minutes.

The equatorial isomer (XXIII)

MP:>220° C. (grad. dec.)

UV λ_(max) (0.01N NaOH) nm (E₁ cm^(1%)): 241 (271), 320 (128), 498(121).

IR ν_(max) (KBr) cm⁻¹ : 3387, 2920, 1730, 1630, 1605.

¹ H NMR (DMSO-d₆) δ:1.15 (3H, d, J=6.0 Hz, 5'-CH₃), 2.31 (3H, s, 3-CH₃),3.90(2H, d, J=5,8 Hz, NH-CH₂ -), 3.94(3H, s, 11-OCH₃), 4.45 (1H, d,J=7.3 Hz, 1"-H), 6.84 (1H, s, 10-H), 7.00 (1H, s, 4-H), 7.21 (1H, s,12-H), 7.91 (1H, s, 7-H).

HPLC*: Retention time 8.6 minutes.

*HPLC: column, Senshu Pak SSC-ODS-262; solvent, CH₃ CN:pH 7

buffer=15:85; flow rate, 2 ml/minute.

Example 3. Preparation of 4'-deamino-4'-hydroxy pradimicin FA-2 (XXVI)

(a) Triethylamine (0.20 ml, 1.43 mmol) was added to a mixture ofpradimicin FA-2 HC1 (150 mg, 0.16 mmol),3,5-di-tert-butyl-1,2-benzoquinone (150 mg, 0.68 mmol) in dry methanol(2.5 ml). The mixture was stirred overnight and concentrated underreduced pressure. To the residue were added ethyl acetate (5 ml) and aq.saturated NaHCO₃ (2 ml), and the mixture was stirred for 30 minutes atroom temperature to precipitate the sodium salt of4'-(3,5-di-t-butyl-2-hydroxy)phenyl imine of pradimicin FA-2 (XXIV, 164mg, 96%).

IR ν_(max) (KBr) cm⁻¹ : 1618, 1259.

UV λ_(max) (methanol) nm (E₁ cm^(1%)): 281 (211), 497 (93).

¹ H NMR (DMSO-d₆) δ:0.95 (3H, d, J=7 Hz, 5'-CH₃), 1.22 (9H, s, t-Bu),1.25 (9H, s, t-Bu), 2.23 (3H, s, 3-CH₃), 4.81 (1H, d, J=8 Hz, 1'-H),5.05 (1H, br)*, 6.42 (1H, d, J=2 Hz, phenyl-H), 6.44 (1H, d, J=2 Hz,phenyl-H), 6.70 (1H, br, 10-H), 6.90 (1H, s, 4-H), 7.10 (1H, br, 12-H),7.40 (1H, br)*, 7.69 (1H, s, 7-H).

*Disappeared upon addition of D₂ O.

(b) A mixture of the product obtained in step (a) (160 mg, 0.15 mmol),formic acid (3 ml) and methanol (3 ml) was heated at 60° C. for 1.5hours. The reaction mixture was concentrated under reduced pressure andthe residue was chromatographed on a column of C18 silica gel (20×200mm). The column was eluted with water and then with 30% acetonitrile.The acetonitrile fractions were checked with HPLC, and the desiredfractions were combined and concentrated to leave an aqueous residuewhich was freeze-dried to give 4'-deamino-4'-oxo pradimicin FA-2 (XXV,105 mg, 83%) as an amorphous powder.

IR ν_(max) (KBr) cm⁻¹ : 1733 (weak), 1607, 1258, 1084.

UV λ_(max) (0.01N NaOH) nm (ε): 318 (14800), 498 (13500).

¹ H NMR (DMSO-d₆) δ:3.94 (3H, s, OCH₃), 6.88 (1H, s, 10-H), 7.25 (1H, s,12-H), 7.95 (1H, s, 7-H).

(c) To a stirred mixture of the product obtained in step (b) (121 mg,0.15 mmol), IN NaOH (0.3 ml), and water (12 ml) was added an aqueoussolution of 0.1M sodium borohydride (0.7 ml) at 5° C. The mixture wasstirred for 1 hour at the same temperature and acidified with 1N H₂ SO₄to destroy the reagent. The mixture was adjusted to pH 8 with NaHCO₃ andchromatographed on a column of C18 silica gel (40×330 mm, 5%acetonitrile) and followed by preparative HPLC (System 500 (Waters), 7%acetonitrile) to give 3 fractions--a faster moving fraction containingthe equatorial isomer, a slower moving fraction containing the axialisomer, and a fraction containing a mixture of both isomers. Eachfraction was concentrated to a small volume, acidified with 1N H₂ SO₄,sand subjected to a short column of C18 silica gel. The column waswashed with water and eluted with 80% acetonitrile. The eluate wasconcentrated to a small volume and lyophilized. The 3 fractions afforded4'-deamino-4'-hydroxy pradimicin FA-2 axial isomer (XXVI, 3 mg, 3%), theequatorial isomer (XXVII, 5.4 mg, 4%), and a mixture thereof.

4'-Deamino-4'-hydroxy pradimicin FA-2 (axial isomer, XXVI)

MP:>220° C. (grad. dec.).

UV λ_(max) (0.01N NaOH) nm (E₁ cm^(1%)): 320 (134), 497 (129).

IR ν_(max) (KBr) cm⁻¹ : 3272, 2917, 1739, 1607.

¹ H NMR (DMSO-d₆) δ:1.10 (3H, d, J=6.4 Hz, 5'-CH₃), 2.34 (3H, s, 3-CH₃),3.69 (1H, dd, J=5.5 & 11.1 Hz, 5"-eq-H), 3.95 (3H, s, 11-OCH₃), 4.40(1H, d, J=6.8 Hz, 1"-H), 4.63 (1H, d, J=7.7 Hz, 1'-H), 6.90 (1H, s,10-H), 7.10 (1H, s, 4-H), 7.27 (1H, s, 12-H), 7.99 (1H, s, 7-H).

HPLC*: Retention time 9.8 minutes.

The equatorial isomer (XXVII)

MP:>220° C. (grad.dec.)

UV λ_(max) (0.01N NaOH) nm (E₁ cm^(1%)): 318 (151), 497 (140).

IR ν_(max) (KBr) cm⁻¹ : 3408, 1733, 1607.

¹ H NMR (DMSO-d₆) δ:1.15 (3H, d, J=6.0 Hz, 5'-CH₃), 2.32 (3H, s, 3-CH₃),3.75 (1H, dd, J=5.1 & 11.1 Hz, 5"-eq-H), 3.94 (3H, s, 11-OCH₃), 4.45(1H, d, J=7.3 Hz, 1"-H), 6.87 (1H, s, 10-H), 7.02 (1H, s, 4-H), 7.24(1H, s, 12-H), 7.93 (1H, s, 7-H).

HPLC*: Retention time 8.5 minutes.

*HPLC conditions same as described in Example 2.

Example 4. Preparation of benanomicin A

(a) The procedure of Example 2, step (a), was followed using pradimicinC HC1 (150 mg, 0.16 mmol) and 3.5-di-t-butyl-1,2-benzoquinone (110 mg,0.5 mmol) to provide the corresponding imine (XXVIII, 212 mg).

IR ν_(max) (KBr) cm⁻¹ : 1622, 1607, 1259, 1080.

UV λ_(max) (MeOH) nm (E₁ cm^(1%)): 288 (259), 478 (98).

¹ H NMR (DMSO-d₆) δ:0.96 (3H, d, J=7 Hz, 5'-CH₃), 1.22 (9H, s t-Bu),1.25 (9H, s, t-Bu), 1.29 (3H, d, J=7 Hz, alanyl-CH₃), 2.22 (3H, s,3-CH₃), 3.90 (3H, s, OCH₃), 4.82 (1H, d, J=8 Hz, 1'-H), 4.94 (1H, br)*,5.09 (1H, br)*, 5.70 (1H, br)*, 5.80 (1H, br)*, 5.98 (1H, s)*, 6.19 (1H,s)*, 6.42 (1H, d, J=2 Hz, phenyl-H), 6.49 (1H, d, J=2 Hz, phenyl-H),6.71 (1H, d, J=2 Hz, 10-H), 6.91 (1H, s, 4-H), 7.13 (1H, d, J=2 Hz,12-H), 7.47 (1H, br)*, 7.68 (1H, s, 7-H), 13.22 (1H, s)*, 14.80 (1H,s)*.

*Disappeared by the addition of D₁ O.

(b) The procedure of Example 2, step (b), was followed using the imineobtained from step (a) above (210 mg, 0.20 mmol) to provide thecorresponding ketone (XXIX, 147 mg 89%).

IR ν_(max) (KBr) cm⁻¹ : 1738 (weak), 1607.

UV λ_(max) (0.02N NaOH) nm (E₁ cm^(1%)): 318 (171), 498 (143).

¹ H NMR (DMSO-d₆) δ:3.96 (3H, s, 11-OCH₃), 6.9 (1H, s, 4-H), 7.32 (1H,s, 12-H).

(c) The procedure of Example 2, step (c), was followed using the ketoneobtained above (80 mg, 0.097 mmol) to provide benanomicin A (II, 8.5 mg,11%), its 4'-equatorial isomer (XXX, 5 mg, 6%) and mixture thereof (5mg).

Benanomicin A (II)

MP:>220° C. (grad. dec.).

UV λ_(max) (NaOH--MeOH) nm (E₁ cm^(1%)): 277 (233), 318 (92), 499 (108).

IR ν_(max) (KBr) cm⁻¹ : 3402, 1733, 1623, 1607.

¹ H NMR (DMSO-d₆) δ:1.12 (3H, d, J=6.4 Hz, 5'-CH₃), 1.33 (3H, d, J=7.3Hz, 17-CH₃), 2.27 (3H, s, 3-CH₃), 3.90 (3H, s, 11-OCH₃), 4.63 (1H, d,J=7.7 Hz, 1'-H), 6.71 (1H, d, J=2.1 Hz, 10-H), 6.94 (1H, s, 4-H), 7.11(1H, d, J=2.1 Hz, 12-H), 7.74 (1H, s, 7-H).

MS (FAB): (Positive) 828 (M+H)⁺, 850 (M+Na)⁺ (Negative) 827 (M)⁻

HPLC*: Retention time 9.5 minutes.

The equatorial isomer (XXX)

MP:>250° C. (grad.dec.).

UV λ_(max) (NaOH--MeOH) nm (E₁ cm^(1%)) 277 (209), 318 (88), 502 (103).

IR ν_(max) (KBr) cm⁻¹ : 3398, 1733, 1627, 1607.

¹ H NMR (DMSO-d₆) δ:1.15 (3H, d, J=6.0 Hz, 5'-CH₃), 1.33 (3H, d, J=7.3Hz, 17-CH₃), 2.29 (3H, s, 3-CH₃), 3.75 (1H, dd, J=5.6 & 11.1 Hz,5"-eq-H), 3.93 (3H, s, 11-OCH₃), 4.45 (1H, d, J=7.3 Hz, 1"-H), 6.79 (1H,s, 10-H), 6.95 (1H, s, 4-H), 7.18 (1H, s, 12-H), 7.84 (1H, s, 7-H).

MS (FAB): (Positive) 828 (M+H)⁺, 850 (M+Na)⁺ (Negative) 826 (M-H)⁻

HPLC*: Retention time 8.8 minutes.

*HPLC: column, Senshu Pak SSC-ODS-262; solvent, CH₃ CN:pH 7buffer=15:85; flow rate, 2 ml/minute.

Compounds Prepared in Examples 1-4

Example 1 ##STR11## Examples 2-4 ##STR12##

What is claimed is:
 1. A compound having the formulawherein R^(a) is H,methyl, or hydroxymethyl, and when R^(a) is methyl or hydroxymethyl, theresulting amino acid has the D-configuration; R^(b) is H or β-D-xylosyl;or a salt thereof; or an ester thereof.
 2. A compound having the formula##STR13## wherein R^(a) and R^(b) are as defined in claim 1; or a saltthereof; or an ester thereof.
 3. A process for preparing a compound ofthe formula ##STR14## wherein R^(a) and R^(b) are as defined in claim 1,or a pharmaceutically acceptable salt thereof, which comprises the stepsof:(a) reacting a compound having the formula ##STR15## wherein R^(a)and R^(b) are as defined above, or a salt thereof, with3,5-di-t-butyl-1,2-benzoquinone in an inert organic solvent to form thecorresponding imine; (b) converting the product of (a) into thecorresponding ketone in the presence of an acid catalyst; (c) reducingthe product of (b) to the corresponding alcohol; and (d) separating theisomers.